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Electrical currents generated by a partially purified Na/Ca exchanger from lobster muscle reconstituted into liposomes and adsorbed on black lipid membranes: Activation by photolysis of caged Ca2+

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Abstract

The Na/Ca exchanger from lobster muscle crossreacts specifically with antibodies raised against the dog heart Na/Ca exchanger. Immunoblots of the lobster muscle and mammalian heart exchangers, following SDS-PAGE, indicate that the invertebrate and mammalian exchangers have similar molecular weights: about 120 kDa. The exchanger from lobster muscle was partially purified and functionally reconstituted into asolectin vesicles which were loaded with 160 mm NaCl. 45Ca uptake by these proteoliposomes was promoted by replacing 160 mm NaCl in the external medium with 160 mm KCl to produce an outwardly-directed Na+ concentration gradient. When the proteoliposomes were adsorbed onto black lipid membranes (BLM), and DMNitrophen-Ca2+ (“caged Ca2+”) was added to the KCl medium, photolytically-evoked Ca2+ concentration jumps elicited transient electric currents. These currents corresponded to positive charge exiting from the proteoliposomes, and were consistent with the Na/Ca exchanger-mediated exit of 3 Na+ in exchange for 1 entering Ca2+. The current was dependent upon the Ca2+ concentration jump, the protein integrity, and the outwardly directed Na+ gradient. KCl-loaded proteoliposomes did not produce any current. Low external Na+ concentrations augmented the current, whereas Na+ concentrations >25 mM reduced the current. The dependence of the current on free Ca2+ was Michaelis-Menten-like, with halfmaximal activation (KM(Ca)) at <10 μm Ca2+. Caged Sr2+ and Ba2+, but not Mg2+, also supported photolysisevoked outward current, as did Ni2+, but not Mn2+. However, Mg2+ and Mn2+ augmented the Cadependent current, perhaps by facilitating the adsorption of proteoliposomes to the BLM. The Ca-dependent current was irreversibly blocked by La3+ (added as 200 μm DMN-La3+). The results indicate that the properties of the Na/Ca exchanger can be studied with these electrophysiological methods.

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Authors and Affiliations

  1. Laboratory of Biophysical Chemistry, Max-Planck-Institute für Biophysik, Kennedyallee 70, D-60596, Frankfurt/M, Germany

    A. Eisenrauch, E. Bamberg & M. P. Blaustein

  2. Department of Physiology, University of Maryland School of Medicine, 655 West Baltimore Street, 21201, Baltimore, MD, USA

    M. Juhaszova & M. P. Blaustein

  3. Department of Physiology, University of Pennsylvania School of Medicine, University Walk, 19104, Philadelphia, PA

    G. C. R. Ellis-Davies & J. H. Kaplan

  4. Department of Biochemistry and Molecular Biology, Oregon Health Sciences University, 3181 SW Sam Jackson Park Road, 97201-3098, Portland, OR

    G. C. R. Ellis-Davies & J. H. Kaplan

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  1. A. Eisenrauch
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  2. M. Juhaszova
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  3. G. C. R. Ellis-Davies
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  4. J. H. Kaplan
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  5. E. Bamberg
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  6. M. P. Blaustein
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The technical assistance of Verena Heiselpetz in some experiments is gratefully acknowledged. This work was partly supported by the Deutsche Forschungsgemeinschaft (SFB 169) and by National Institutes of Health grants HL30315 and GM39500 to JHK and HL45215 and NS16106 to MPB. MPB was the recipient of a Senior Scientist Award from the Alexander von Humboldt Stiftung.

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Eisenrauch, A., Juhaszova, M., Ellis-Davies, G.C.R. et al. Electrical currents generated by a partially purified Na/Ca exchanger from lobster muscle reconstituted into liposomes and adsorbed on black lipid membranes: Activation by photolysis of caged Ca2+ . J. Membarin Biol. 145, 151–164 (1995). https://doi.org/10.1007/BF00237373

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  • DOI: https://doi.org/10.1007/BF00237373

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